The appropriate detector and effective procedure of sample pretreatment enabling the elimination of matrix effects in analysis of complex samples are significant elements of each high-performance separation method of analysis. The application of biosensors in high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) could improve detection and, in many cases, allows detection of solutes otherwise undetectable by common method. For this purpose, a homogeneous derivatization can be employed, involving flow-through reactors with immobilized biomolecules or integrated enzymatic sensors or immunosensors. In gas chromatography (GC), two areas where biosensing is employed are olfactometry and antennography. In CE, numerous applications have been developed using on-line immunoassay and detection with single cells.